The human herpesviruses cause a spectrum of clinically significant acute and life-long latent infections which can be life-threatening in immunocompromised individuals. Viral DNA replication is pivotal in the dual life cycles of all herpesviruses in that the absence of DNA replication is a hallmark of viability in latently infected cells, whereas the onset of DNA replication signals either the commitment of acutely infected cells to ultimate lysis, or the reactivation of latent virus. Using herpes simplex virus type I (HSV-1) as a model system, the objectives of this proposal are to elucidate the roles of the two HSV-1 origins of replication, oriL and oriS, and the proteins that bind to them in origin-dependent DNA replication. Specifically, viral mutants defective in the synthesis of two HSV-1-encoded origin-binding proteins, OBP and OBPC, will be isolated and characterized in order to identify their unique roles in viral DNA replication. The interactions of viral origin-binding proteins OBP and OBPC and the cellular protein, OF-1, with each other, with HSV-1 origins, and with other HSV-1 DNA replication proteins will be examined. The significance of these interactions for origin-dependent DNA replication and for transcription of origin-flanking genes will be assessed in transient assays and in the context of viral infection. Collectively, these studies will identify the roles of specific origin-associated DNA sequences and the proteins that bind to them in HSV-1 origin-dependent DNA replication. Efforts will also be made to determine the molecular basis for the recent observation that glucocorticoids enhance DNA replication from oriL, and nerve growth factor (NGF) represses DNA replication from oriS in NGF-differentiated neural cells. These studies should help to establish the functional significance of the existence of two types of origin in HSV-1. A better understanding of the mechanisms involved in HSV-1 origin- dependent DNA replication should shed light on the disease-producing properties of herpesviruses and may ultimately provide novel approaches to intervention in the herpesvirus life cycle.